Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme and a single-chain Fv antibody that targets CCR5.

The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have reduced or absent CCR5 are apparently otherwise healthy, but are resistant to HIV infection. With the goal of reducing CCR5 and protecting CCR5+ cells from R5-tropic HIV, we used Tag-deleted SV40-derived vectors to deliver several anti-CCR5 transgenes: 2C7, a single-chain Fv (SFv) antibody; VCKA1, a hammerhead ribozyme; and two natural CCR5 ligands, MIP-1alpha and MIP-1beta, modified to direct these chemokines, and hence their receptor to the endoplasmic reticulum. These transgenes were delivered using recombinant, Tag-deleted SV40-derived vectors to human CCR5+ cell lines and primary cells: monocyte-derived macrophages and brain microglia. All transgenes except MIP-1alpha decreased CCR5, as assayed by immunostaining, Northern blotting, and cytofluorimetry (FACS). Individually, all transgenes except MIP-1alpha protected from low challenge doses of HIV. At higher dose HIV challenges, protection provided by all transgenes diminished, the SFv and the ribozyme being most potent. Vectors carrying these two transgenes were used sequentially to deliver combination anti-CCR5 genetic therapy. This approach gave approximately additive reduction in CCR5, as measured by FACS and protected from higher dose HIV challenges. Reducing cell membrane CCR5 using anti-CCR5 transgenes, alone or in combinations, may therefore provide a degree of protection from R5-tropic strains of HIV.

Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4.

RNA interference (RNAi) is an evolutionarily conserved process by which plants and animals protect their genomes utilizing small, double-stranded RNAs to degrade target RNAs in a sequence-specific manner. Post-transcriptional gene silencing by these moieties can lead to degradation of both cellular and viral RNAs. It has recently been shown that double-stranded, small interfering RNAs (siRNAs) of 21-25 nucleotides can be transfected into relevant cells to target specific RNAs. This approach was utilized to inhibit human immunodeficiency virus type I (HIV-1) infection in human cells. siRNAs with homology to a motif in the mRNA that encodes for the HIV-1 chemokine coreceptor CXCR4 was utilized. Complementary studies via immunofluorescence microscopy and fluorescence-activated cell sorting demonstrated downregulation of CXCR4 from the surface of cells transfected with the specific siRNAs. As well, siRNAs without sequence homology to CXCR4 were used as controls and demonstrated no downregulation of CXCR4. siRNAs targeted to another chemokine coreceptor, APJ, showed specificity for downregulation of APJ but had no effects on CXCR4. Transfections with siRNAs targeting CXCR4 mRNA were shown to inhibit HIV-1 envelope fusion, which is relatively resistant to most viral inhibitors targeting chemokine coreceptors. The specificity of this effect was demonstrated by the inhibition of fusion by CXCR4-tropic and dual-tropic (CXCR4 and CCR5) envelope glycoproteins from HIV-1 on CXCR4+ indicator cells, but the lack of effects by siRNAs targeting CXCR4 mRNA on dual-tropic HIV-1 envelopes in CCR5+ indicator cells utilizing these fusion assays. Interestingly, siRNAs targeting CXCR4 selectively inhibited CXCR4-tropic cell-free virus infection of human cells but at only modest levels as compared to cell:cell fusion. siRNA may be a potential molecular therapeutic approach to alter a cellular cofactor critical for infection of human cells by relevant strains of HIV-1. The targeting of a cellular cofactor, rather than the HIV-1-specific mRNAs or genomic RNA, holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi directly against this virus.

Reduction of serum melatonin levels in HIV-1-infected individuals' parallel disease progression: correlation with serum interleukin-12 levels.

BACKGROUND: During the natural history of human immunodeficiency virus type I (HIV-1) infection, an impairment of interleukin-12 (IL-12) production precedes a switch from a T-helper 1 (Th1) to a T-helper 2 (Th2) stage of cellular immunity. Melatonin, the main hormone produced by the pineal gland, seems to promote a Th1 response by increasing the production of IL-12 in vitro. The aim of this study was to measure and correlate serum levels of melatonin and IL-12 in a cohort of HIV-1-infected individuals. PATIENTS AND METHODS: 77 anti-HIV-1-positive subjects were enrolled: 20 were in CDC stage A, 25 in CDC stage B and 32 in CDC stage C. 30 healthy HIV-1-seronegative subjects were recruited as controls. IL-12 and melatonin concentrations were quantitated in serum samples. RESULTS: Mean levels of serum melatonin were significantly lower in HIV-1-infected individuals in comparison with controls (p < 0.001). Within the HIV-1-seropositive group, mean melatonin and IL-12 concentrations were significantly lower in patients in CDC stage C, as compared with patients in CDC stages B and A (p < 0.01). CONCLUSION: During the natural history of HIV-1 disease, serum melatonin levels are progressively reduced. This reduction may be related to the impairment of Th1 immunoresponses.

Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART.

Prostratin is a unique phorbol ester that stimulates protein kinase C activity but is nontumor promoting. Remarkably, prostratin is also able to inhibit de novo human immunodeficiency virus type 1 (HIV-1) infection yet up-regulate viral expression from latent proviruses. Prostratin's lack of tumor promotion, coupled with its ability to block viral spread yet induce latent proviral expression, prompted studies to determine whether this compound could serve as an inductive adjuvant therapy for patients treated with highly active antiretroviral therapy (HAART). The current experiments indicate that prostratin is a potent mitogen for mononuclear phagocytes possessing many of the activities of phorbol myristate acetate (PMA) with notable functional differences. Prostratin, like PMA, accelerates differentiation of the myeloid cell-lines, HL-60 and THP-1, as well as mononuclear phagocytes from bone marrow and peripheral blood. Enzyme-linked immunosorbent assay and gene array analyses indicate significant changes in the expression of proteins and messenger RNA after treatment of cells with prostratin, consistent with phagocyte activation and differentiation. Prostratin blocks HIV-1 infection relating to down-regulation of CD4 receptor expression. The array analysis indicates a similar down-regulation of the HIV-1 coreceptors, CXCR4 and CCR5, and this may also reduce viral infectivity of treated host cells. Finally, prostratin is capable of up-regulating HIV-1 expression from CD8+ T lymphocyte-depleted peripheral blood mononuclear cells of patients undergoing HAART. This novel observation suggests the agent may be an excellent candidate to augment HAART by inducing expression of latent HIV-1 with the ultimate goal of eliminating persistent viral reservoirs in certain individuals infected with HIV-1.

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies.

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.

Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector.

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.

SV40-derived vectors provide effective transgene expression and inhibition of HIV-1 using constitutive, conditional,and pol III promoters.

Vectors based on recombinant SV40 viruses (rSV40) are highly effective in delivering transgene expression driven by constitutive promoters. We tested here whether these vectors could be used with conditional promoters and promoters using RNA polymerase III transcription, with inhibition of HIV-1 by Tat activation response (TAR) decoys as a functional measure of effective transgene delivery and activity. TAR decoys inhibit HIV-1 Tat, a trans-activator of HIV-1 transcription. Tat acts early in the viral replicative cycle and is essential for efficient viral replication. We evaluated rSV40 gene delivery using two different inhibitors of Tat. One was a dual function polyTAR gene encoding 25 sequential TAR elements (TAR(25)), plus an antisense tat, driven either by HIV-1 long terminal repeat (HIV-LTR) as a conditional promoter, or by cytomegalovirus immediate-early promoter (CMV-IEP) as a constitutive promoter. The other inhibitor was a single TAR decoy, driven by the U6 small nuclear RNA promoter (U6-P). These decoys were delivered to unselected cells in two different human T lymphocyte lines and to unstimulated primary human peripheral blood mononuclear cells (pbmc). Gene delivery was confirmed by PCR, and expression by RT-PCR. By in situ hybridization analysis, >95% of cells were transduced. These transgene constructs protected all cell types tested from HIV-1, as measured by syncytia formation and p24 antigen release. Somewhat better inhibition of HIV-1 replication was achieved with HIV-1 long terminal repeat (HIV-1 LTR) as a conditional promoter than with the constitutive CMV-IEP. The U6-P was also very effective, driving a TAR(1) transcript. Cell viability was not detectably affected by TAR decoy expression. Thus, rSV40 vectors effectively deliver HIV-1-inhibitory RNAs using either constitutive or conditional pol II promoters, or using a pol III promoter. The versatility of this gene delivery system may prove to be useful in anti-HIV-1 therapeutics.

Association of provider and patient characteristics with HIV-infected women's antiretroviral therapy regimen.

OBJECTIVE: We explored the effect of patient and provider factors on the type of antiretroviral regimen among women receiving therapy. PATIENTS: Five hundred ninety-five New York State nonpregnant HIV+ women with full Medicaid eligibility and at least 1 month of a prescribed antiretroviral regimen in federal fiscal years (FFY) 1997-1998 and intervals in FFY 1997-1998, who had delivered a liveborn baby within 5 years. MEASUREMENTS: From pharmacy claims in 4 6-month intervals in FFY 1997-1998, data were extracted on (1) an acceptable > or = 2 antiretroviral combination regimen per expert guidelines; and (2) a highly active regimen, including a protease inhibitor or nonnucleoside analog (highly active antiretroviral therapy [HAART]). RESULTS: Of 1514 woman-6-month intervals with filled antiretroviral prescriptions, 82% had an acceptable regimen, and of 1246 woman-6-month intervals on acceptable antiretroviral therapy, half demonstrated the use of HAART. Adjusted odds ratios (AORs) of acceptable antiretroviral therapy were higher (p < .05) for HIV specialty care (AOR = 1.71 for one or two visits; AOR = 2.10 for 3+ visits) or HIV clinical trials site care (AOR = 1.43; 95% confidence interval [CI]: 1.01, 2.04). Among women on acceptable antiretroviral regimens, those aged older than 25 years (AOR = 1.69; CI: 1.13, 2.53) or who were high school graduates (AOR = 1.50; CI: 1.09, 2.06) had higher odds of HAART. Methadone-treated women had twofold and nearly threefold higher AORs of acceptable antiretroviral regimens and HAART, respectively, than current drug users. CONCLUSION: Provider HIV expertise is associated with receipt of an acceptable antiretroviral regimen in women, although receipt of HAART is affected more by age, education, and current drug abuse. Methadone treatment seems to improve access to acceptable antiretroviral regimens as well as to HAART.

Human immunodeficiency virus type 1-infected persons with residual disease and virus reservoirs on suppressive highly active antiretroviral therapy can be stratified into relevant virologic and immunologic subgroups.

A significant percentage of human immunodeficiency virus type 1 (HIV-1)-infected persons treated with highly active antiretroviral therapy (HAART) will develop plasma HIV-1-specific virion RNA levels <50 copies/mL. HIV-1-infected persons receiving virally suppressive HAART were studied with a viral outgrowth assay of the patients' peripheral blood mononuclear cells (PBMC), and a quantitative polymerase chain reaction assay was used to analyze HIV-1 2-long terminal repeat (2-LTR) circular DNA in PBMC, which indicates new HIV-1 infections of cells in vivo. Viral outgrowth in vitro correlated inversely with the level of peripheral blood CD4(+) T lymphocytes. Detection and quantitation of 2-LTR circular DNA correlated strongly with viral outgrowth patterns and inversely with CD4(+) T lymphocyte counts. Relevant subgroups of HIV-1-infected subjects on suppressive HAART with residual viral disease and reservoirs can now be stratified.

Inhibition of HIV-1 infection by down-regulation of the CXCR4 co-receptor using an intracellular single chain variable fragment against CXCR4.

CXCR4 is the major co-receptor used by X4 strains of human immunodeficiency virus type I (HIV-1). In HIV-1-infected patients, the appearance of X4 strains (T cell line-tropic) correlates with disease progression. Since its discovery, the CXCR4 co-receptor has been a major target for different agents which block its function, such as stromal-derived factor 1alpha (SDF-1alpha) and the anti-CXCR4 monoclonal antibody, 12G5. In the present studies, the 12G5 hybridoma was used to construct a single-chain variable antibody fragment (SFv). Murine leukemia virus (MLV) and simian virus 40 (SV(40)) were utilized as delivery vehicles for the anti-CXCR4 SFv. Intracellular expression of the anti-CXCR4 SFv led to down-regulation of this critical co-receptor, as demonstrated by immunostaining. This effect significantly and specifically protected transduced cells from challenge with HIV-1, as measured by HIV-1 p24 antigen expression. Inhibition of HIV-1 replication was specific for X4 HIV-1 strains as demonstrated by MAGI assays. HeLa-CD4/betagal-CCR5 cells expressing the anti-CXCR4 SFv showed significant inhibition of infectivity by the X4 HIV-1 strain NL4-3, but not with the R5 HIV-1 strain Bal. Thus, this anti-HIV-1 molecular therapy has the potential to inhibit HIV-1 replication and virion spread. Targeting CXCR4 by intracellular immunization could be of additional benefit to certain HIV-1-infected patients on highly active antiretroviral therapy (HAART).

Rabies virus-based vectors expressing human immunodeficiency virus type 1 (HIV-1) envelope protein induce a strong, cross-reactive cytotoxic T-lymphocyte response against envelope proteins from different HIV-1 isolates.

Novel viral vectors that are able to induce both strong and long-lasting immune responses may be required as effective vaccines for human immunodeficiency virus type 1 (HIV-1) infection. Our previous experiments with a replication-competent vaccine strain-based rabies virus (RV) expressing HIV-1 envelope protein from a laboratory-adapted HIV-1 strain (NL4-3) and a primary HIV-1 isolate (89.6) showed that RV-based vectors are excellent for B-cell priming. Here we report that cytotoxic T-lymphocyte (CTL) responses against HIV-1 gp160 are induced by recombinant RVs. Our results indicated that a single inoculation of mice with an RV expressing HIV-1 gp160 induced a solid and long-lasting memory CTL response specific for HIV-1 envelope protein. Moreover, CTLs from immunized mice were not restricted to the homologous HIV-1 envelope protein and were able to cross-kill target cells expressing HIV-1 gp160 from heterologous HIV-1 strains. These studies further suggest promise for RV-based vectors to elicit a persistent immune response against HIV-1 and their potential utility as efficacious anti-HIV-1 vaccines.

Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy.

Residual HIV-1 disease remains in the vast majority of patients treated with even the most intensive highly active antiretroviral therapy (HAART). There are at least two well-described molecular mechanisms for HIV-1 persistence in these patients. These include proviral latency in resting CD4+ T-cells, as well as 'cryptic' residual viral replication. As well, potential sanctuary sites, including the brain and testes, may be important areas which will hinder HIV-1 eradication attempts. It is not clear whether other sites of HIV-1 persistence, including tissue-bound infected monocytes/macrophages, may also be involved in residual HIV-1 disease during virally-suppressive HAART.

Retroviruses and opportunistic infections--eighth annual conference.

The major focus of the Eighth Annual Retrovirus Conference was recent developments in the field of human retrovirology and related opportunistic infections at the forefront of clinical and scientific research. A number of state of the art lectures and symposia were followed by evening poster sessions. Viral reservoirs were particularly highlighted in the meeting with major focus on the brain as a site of HIV-1 persistence. Highly active antiretroviral therapy (HAART) was discussed in detail as well as optimism for future antiretrovirals, such as entry and fusion inhibitors. Data were also presented on co-receptor inhibitors and their potential as future HIV medications.